To date, no single therapeutic approach has proven universally effective in preventing SpA or ameliorating SpA. Therefore, there is a need for compositions and methods that may be used safely and effectively to prevent or treat SpA in a variety of different contexts. The invention applies to human and veterinary applications.
The Spondyloarthropathies (SpA) are an important group of chronic inflammatory disorders, affecting both the axial and peripheral skeleton. Within the SpA group, several entities are recognized: Ankylosing Spondylitis (AS), Psoriatic Arthritis (PsA), Enteropathic Arthritis or Arthritis associated with inflammatory bowel disease (IBD-SpA), Reactive Arthritis (ReA) including Reiter's syndrome and the undifferentiated forms (UspA) and, possibly, also Whipple disease and Behcet disease. (John H. Klippel, et al., Rheumatology Mosby Saint Louis (Ill.) Publ Jaar: 1994 Ed. by John H. Klippel and Paul A. Dieppe.)
These diseases can be easily classified into the SpA group since they display a number of striking similarities: enthesitis, strong genetic predisposition in HLA-B27 positive individuals, involvement of the sacroiliacal and intervertebral joints, asymmetrical oligoarthritis in the peripheral joints mainly affecting the lower limbs. Enthesitis, leading to neochondro- or neo-osteogenesis and joint ankylosis, is the hallmark feature of all forms of SpA affecting synovial joints (e.g. knee joint), cartilaginous joints (e.g. discovertebral junction), syndesmoses (e.g. upper part of the sacroiliacal joints) and extra-articular enthesis (e.g. insertion of the Achilles tendon) (Ball J., Ann Rheum Dis. 1971 May; 30(3):213-23; Benjamin M. and McGonagle D., J Anatomy 2001; 199(5):503-526).
A therapy aimed at disrupting the enthesial process of proliferation, differentiation, cartilage and bone formation leading to ankylosis, is currently lacking. The actual treatment is solely aimed at controlling the inflammatory process by using steroids, non-steroidal anti-inflammatory drugs and second-line antirheumatic immunomodulatory drugs, all with limited success and/or considerable side-effects without specific interference with the process of cartilage and bone formation. Presently, for end stage refractory diseases, surgical management in the form of for example hip replacement is advised. Therefore the need for specific treatments of ankylosing enthesitis remains high.
The present invention embodies that specific morphogens such as Bone Morphogenetic Protein (BMPs) including BMP-2, BMP-6 and BMP-7 play a key role in steering the pathological process leading to joint ankylosis. There are now known to be at least 15 members of the family of the BMP's; all except BMP-1 have been classified as members of the transforming growth factor-β (TGF-β) superfamily. The TGF-β superfamily is a large group of structurally related polypeptides, capable of regulating a wide array of cellular processes such as proliferation, differentiation, lineage determination, motility, adhesion and death. BMP-7 has previously been shown to possess anti-inflammatory properties (Vukicevic, S. et al., Journal of Clinical Investigation 1998; 102 (1): 202-214; Maric, I. et al. Abstract 4th International Conference on BMPs, Oct. 17-21, 2002, Sacramento, Calif.).
The invention therefore embodies the specific treatment of SpA or SpA enthesitis by using inhibitors of BMPs, or related family members, such as Noggin, Chordin, Gremlin and Follistatin, or cellular therapies directed against these specific morphogens. Treatment aimed at blocking these morphogens can be both locally administered as well as systemically. Chordin, for instance, binds to BMP-2 and BMP-4 in the extracellular space, blocking the interaction of BMPs with their receptor.
Another embodiment of the present invention relates to the use of molecules that can neutralize the activity of BMPs by interfering with its synthesis, formation, translation, transcription, receptor binding and/or receptor binding mediated signal transduction, for the treatment or prevention of SpA or SpA enthesitis.
A strikingly simple signaling pathway is used by BMPs and related factors (for review see Massague, J. Annu Rev. Biochem. 1998; 67: 753-791; Massague, J. Nat. Rev. Mol. Cell. Biol. 2000; 1:169-178). The enormous diversity of cellular responses is achieved by regulatory mechanisms on all levels of this pathway. The dimeric ligand assembles a receptor complex consisting of two different receptor serine/threonine kinases (type I and type II receptors). The type II receptor phosphorylates the type I receptor, resulting in the activation of this receptor kinase. The type I receptor subsequently phosphorylates specific receptor substrates (Smad molecules in vertebrates, Mad in Drosophila). The phosphorylated receptor-Smad (R-Smad) associates with common Smad-4 and translocates to the nucleus. The Smad complexes have DNA binding and transcriptional activity.
Vertebrate type I receptors can be divided into three groups. One group includes the TGFβ type I receptor (TβRI), formerly known as Activin-receptor like kinase-5 (ALK-5), Activin-receptor type Ib (ActRIb) or ALK-4 and ALK-7. The second group includes BMPRIa (ALK-3) and BMPRIb (ALK-6). The third group includes ALK-1 and ActRIa (ALK-2). Similar type I receptors are found in invertebrates: In vertebrates the type II receptors include TβRII, BMPRII, ActRIIa and ActRIIb.
Regarding the binding of BMPs with recptors, ActRI is binding BMP-2, BMP-6 and BMP-7 (Ebisawa et al. J. Cell. Sci. 1999; 112: 3519-3527) with BMPRII, and ActA, BMP-6 and BMP-7 with ActRII. BMPRIa is binding BMP-2 with ActRIIB. BMP-2, -4 and -7 are binding BMPRIb and BMPRII.
Most ligands interact with the receptors as homodimers. However, the existence of TGFβ (Ogawa, Y. et al. J. Biol. Chem. 1992; 267: 2325-2328) and Activin heterodimers (Ling, N. et al. Nature 1986; 321: 779-782) has been documented. BMP heterodimers have been constructed in vitro (Aono, A. et al. Biochem. Biophys. Res. Commun. 1995; 210: 670-677) but have not been demonstrated in vivo. TGFβ and Act type I receptors only bind ligands bound to type II receptors (Laiho, M. et al. J. Biol. Chem. 1990; 265: 18518-18524), BMP type I and type II receptors bind ligand with high affinity when expressed together and with low affinity when expressed separately (Liu, F. et al. Mol. Cell Biol. 1995; 15: 3479-3486).
The BMP receptors use Smad1, Smad5 and Smad8 as substrates and specific DNA-binding co-factors (FAST, OAZ, Mixer and Milk), as well as other transcription factors such as AP-1 and AML proteins (Massague, J. et al. Genes Dev. 2000; 14: 627-644) and additional co-activators (p300/CBp) or co-repressors (TGIF, c-Ski and SnoN) for interaction with genes.
FKBP12 is a cytosolic protein enhancing cytosolic calcium release and inhibiting TGFβ type I receptors by blocking TGFβ type II induced type I receptor phosphorylation (Chen, Y. G. et al. EMBO J. 1997; 16: 3866-3876). BMP receptor associated molecule-1 (BRAM1) may have a similar function on BMPRIa (Kurozumi, K. et al. Genes Cells 1998; 3: 257-264). BAMBI (BMP and activin membrane-bound inhibitor) was identified as a BMP inhibitor in Xenopus, hindering BMP—receptor heterodimerisation (Onichtchouk, D. et al. Nature 1999; 401: 480-485). A human homologue nma has also been identified (Wordinger, R. J. et al. Mol. Vis. 2002; 8: 241-250). Screening has identified several other receptor interacting proteins such as TRIP-1, STRAP, PP2A and TRAP-1, but their precise functions remains to be elucidated.
Inhibitors, antagonists, receptor and Smad modulation, as well as inhibitory Smads (Smad6 and Smad7) modulate BMP signaling pathways. Many antagonists of BMP signal transduction are already known in the art and they include Fetuin glycoprotein, also known as α2-HS glycoprotein in humans, Noggin and Chordin. Noggin, a 32 kD glycoprotein (Smith, W. C. et al. Cell 1992; 70: 829-840) and Chordin, a 120 kD molecule related to thrombospondin-1 (Francois, V. et al. Cell 1995; 80: 19-20), are secreted proteins specifically antagonizing BMP activity by blocking BMP interaction with cell surface receptors. Short gastrulation (Sog) is the Drosophila homologue of Chordin.nhibiting Dpp (protein encoded by the decapentaplegic gene). Noggin displays specificity, characterised in that binding is very tight to BMP-2 and BMP-4 (Kd=2×10−11 M) and weaker to BMP-5, BMP-6 and BMP-7. Fetuin has been shown to block osteogenesis, a function promoted by BMP, in a culture of rat bone marrow cells and that a Fetuin derived peptide binds BMP 2 (M. Demetriou et al., J. Biol. Chem. 1996; 271:12755-61.) The DAN family of BMP antagonists, containing vertebrate DAN, Dante, Gremlin, Cerl and ‘Protein Related to DAN and Cerberus’ (PRDC), as well as Xenopus Cerberus, Chick caronte and C. elegans CdCanl, also prevent interaction of BMPs with signaling receptors (Hsu, D. R. et al. Mol. Cell. 1998; 1: 673-683). Follistatin is a secreted glycoprotein that antagonizes Activin, but that also binds some BMPs (Iemura, S. et al. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 9337-9342). Latency-associated protein is in fact the TGFβ propeptide non-covalently bound to the mature polypeptide inhibiting TGFβ receptor interaction (McMahon, G. A. et al. Biochem. J. 1996; 313: 343-351).